A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.
A gas chromatograph equipped with a split/splitless injector (splitless mode 250°C, injection volume 1μ) interfaced to a mass spectrometer operated in chemical ionization mode (CI) should be used for the analysis). Compounds are separated on HP-1MS column (30m x 0.25mm x 025μm) held at 40°C for 1min after injection, and then temperature programmed at 15°C/min to 250°C (for 10min), with helium as the carrier gas (constant flow 0.7ml/min). For the MS-based analysis both, a quadropole (e.g. Agilent HP5973) and an ion trap-based (e.g. Varian Saturn 2200) mass spectrometer can be used. As a CI gas either isobutane (comes in gas tanks) or methanol (vapors from a liquid reservoir are used) can be use, however, in each case the fragments of the ionization reaction should be checked before the selected ion count (SIC) programs are created. We have used the following program for a Varian GC 3900/Saturn MS 2200 system and methanol as the CI reagent: 5.00-11.30min ions (M+1)+ 153-158 (methyl salicylate 153, methyl 2H5-salicylate); 11.30-13.30min ions (M+1)+ 207-228 (methyl jasmonate 207 and 225, methyl dihydrojasmonate 209 and 227); 13.30-16.30min ions (M+1)+ 237-300 (methyl abscisic acid 261, methyl 2H6-abscisic acid 267 (abscisic acid produces predominantly [M-H2O+1]+ ions). All compounds should be identified by comparison with authentic commercially available standards. Quantification of JA, SA, and ABA is done by correlating the peak area (extracted ions) with the peak area (extracted ions) of the respective internal standard and is also based on the fresh weight of the plant material used.
The method presented here has been proven to work reliably for a wide array of plant signaling compounds provided that the size and the chemical nature of the compound does not prevent it from being extracted, methylated, and vaporized. Also, the methylation procedure as it is described herein is not the only way of derivatization. Silyllation and acetylation are alternative methods and protocols can be easily found online.
If the mass spectrometer is not capable of single ion monitoring the mass range that is recorded during a run should be limited to exclude too many unwanted ions from the process. For example, jasmonic acid (JA) and its internal standard dihydro jasmonic acid (dhJA) produce two major (M+1)+ ions, 207 and 225 for JA, and 209 and 227 for dhJA during chemical ionization with methanol. Because these two major (M+1)+ masses (207 and 225 for JA; 209 and 227 for dhJA) do not have the same abundance rationfor JA and dhJA, they should both be extracted and used for quantification. Therefore, a mass range from 207 to 228 should be recorded and the specific ions should be extracted later.